Substrate recognition by Clp chaperones is dependent on interactions with motifs composed of specific peptide sequences. We studied the binding of short motif-bearing peptides to ClpA, the chaperone component of the ATP-dependent ClpAP protease of E. coli in the presence of ATPgammaS and Mg(II) at pH 7.5. Binding was measured by isothermal titration calorimetry (ITC) using the peptide, AANDENYALAA, which corresponds to the SsrA degradation motif found at the C-terminus of abnormal nascent polypeptides in vivo. One SsrA peptide was bound per hexameric ring of ClpA with an association constant of 5 E+6 1/M. Binding was also assayed by changes in fluorescence of an N-terminal dansylated SsrA peptide, which bound with the same stoichiometry of one per ClpA hexamer (association constant of 1 E+7 1/M). Similar results were obtained when ATP was substituted for ATPgammaS at 6 C. Two additional peptides, derived from the phage P1 RepA protein and the E. coli HemA protein, which bear different substrate motifs, were competitive inhibitors of SsrA binding and bound to ClpA hexamer with the apparent association constant >3 E+7 1/M. DNS-SsrA bound with only slightly reduced affinity to deletion mutants of ClpA missing either the N-terminal domain or the C-terminal nucleotide-binding domain, indicating that the binding site for SsrA lies within the N-terminal nucleotide-binding domain. Because only one protein at a time can be unfolded and translocated by ClpA hexamers, restricting the number of peptides initially bound will help to avoid non-productive binding of substrates and aggregation of partially processed proteins. The Ca(II)-binding protein calmyrin is believed to be an important signaling protein. This protein contains four EF-hand motives, two of which bind Ca(II) with high affinities. Calmyrin is expressed in a variety of human tissues and interacts with a broad spectrum of proteins, yet the mechanisms used by this single protein to transmit Ca(II) signals to such a variety of targets remains unknown. One possible Ca(II)-induced mechanism involves exposure of hydrophobic pockets that bind to alpha-helices of a protein. Second potential mechanism involves a Ca(II)-myristoyl switch that leads to the anchoring of Ca(II)-binding proteins to intracellular or plasma membranes. Calmyrin has been shown to be myristoylated at its N-terminus, but thus far Ca(II)-dependent translocation to intracellular membranes has not been demonstrated. A third mechanism may be related to a Ca(II) dependent oligomerization. To establish a possible role of oligomerization in Ca(II)-signal transduction, we characterized oligomerization of the recombinant calmyrin and identified biologically active calmyrin forms. Non-reducing SDS/PAGE showed that in vitro apo- and Ca(II)-bound calmyrin oligomerizes, forming stable intermolecular disulfide bridges. Ultracentrifugation under reducing conditions indicated that apo-calmyrin exists in an equilibrium of a 21.9 kDa monomer and a 43.8 kDa dimer, with dimerization constant of 1.8 E+3 1/M at 6 C. This result was confirmed by gel filtration of apo- and Ca(II)-bound calmyrin. Importantly, both monomer and dimer underwent significant conformational changes in response to binding of Ca(II). However, when cell extracts were analyzed under non-reducing conditions by Western blotting, only monomeric calmyrin was detected in human platelets and lymphocytes, and in rat brains. Moreover, in contrast to the recombinant calmyrin, crosslinking experiments were unable to detect any dimeric species of calmyrin regardless of Ca(II) concentrations. In summary, our current data indicate that although calmyrin forms stable covalent dimmers in vitro, it appears to function as a monomer in vivo.